Heparin and other natural and artificial acid polysaccharides or mucosaccharides are polyanions which exhibit anticoagulant properties, and are often used therapeutically to reduce or prevent coagulation of a patient's blood.
Blood coagulation or clotting is the precipitation of fibrin from blood or plasma, and depends, in part, on a complex cascade of plasma proteins (factors). Some of these blood coagulation factors are routinely used as an indicator of a patient's clinical condition or of a patient's response to therapeutic drugs.
The primary coagulation assays, prothrombin times (PT) and activated partial thromboplastin times (aPTT), measure the amount of time required for the formation of the first filament of fibrin. The presence of heparin, however, artificially increases the PT and aPTT, thus reducing the accuracy of the coagulation assays, thereby providing a false indication of the patient's clinical condition. It is, therefore, very desirable to determine the accurate coagulation parameters of a patient's blood independent of therapeutic or incidental heparin levels. In order to do this, heparin in the sample of the patient's blood must be efficiently removed (or neutralized), before the coagulation parameters are assayed. Practitioners routinely remove or identify heparin using methods such as protamine neutralization, resin absorption, or thrombin/reptilase times, but these methods may be time consuming, cumbersome, and expensive.
In protamine neutralization, for example, a precisely determined dosage of protamine sulfate (a low molecular weight fish polypeptide extract) is typically used. The dosage is generally calculated based on a ratio of one mg of protamine neutralizing 90 units of lung heparin or 115 units of intestinal heparin. In cardiothoracic surgery, for example, 150-300 mg of protamine may be required. Determining the proper dosage requires the utmost precision because protamine, and other neutralizing agents such as polybrene, are soluble in plasma. Any excess neutralizing agent not combined with heparin will remain in the plasma and may interfere with the coagulation test.
Furthermore, the number and noxiousness of protamine sulfate's side effects exacerbates the inherent risks associated with its use, and may contribute to a patient's morbidity and/or mortality. Common side effects include decreased peripheral vascular resistance, vasodilation, hypotension, decreased cardiac output, dyspnea, increased or decreased pulmonary arterial resistance, decreased arterial PO.sub.2, bleeding, or complement activation (via-the heparin-protamine complex). In patients connected to an extracorporeal circuit, the major adverse reactions may include immediate anaphylactic shock (an antibody-mediated allergic reaction which is not dose-dependent) or a delayed response characterized by noncardiogenic pulmonary edema or persistent hypotension (which probably is dose dependent).
An alternative to protamine neutralization is the removal of heparin from the blood, typically by the addition of a heparin complexing agent. Two means for removing heparin from blood plasma are available commercially. One, the Probe-Tek Heparin Adsorbent, manufactured by Probe-Tek, Inc., is a cationic modified cellulose which is supplied as a premeasured dose of heparin adsorbent in a tube, to which 1 ml blood plasma is added. After gently mixing and standing for one minute, the plasma with the adsorbent is centrifuged at 1500 times the force of gravity for 10 minutes. The supernatant is then carefully removed from the tube with a pipette, and used for testing.
The second means, Hepasorb.RTM., is manufactured by Organon Teknika Corporation and is a cellulose which has been modified to contain quaternary ammonium groups. Hepasorb (70 mg) is added to a test tube, and is agitated gently with 1 ml blood plasma for about 10 seconds. It is cautioned that prolonged agitation can cause denaturation of the plasma proteins. After agitation the tube must be mixed slowly for 10 minutes at room temperature and centrifuged for 5 minutes at 12000 times the force of gravity. The supernatant is then withdrawn from the centrifuge tube for testing.
All of these methods and products for removing heparin from a blood sample are difficult and time-consuming, require specialized equipment, require about 20 minutes for even an experienced laboratory worker to prepare a single sample of plasma for testing, require extreme care in the removal of the supernatant plasma after centrifuging to prevent contamination of the deheparinized plasma with adsorbent, and do not completely solve the need articulated by practitioners in the art.
For example, during surgery, the majority of deleterious reactions due to the presence of protamine occur immediately (i.e., they are not dose dependent). A filter for removing substantially all of the heparin typically used (about 15,000-30,000 units) would be most desirable, particularly if the filter could achieve a flow rate of about 2.5-6 liters/minute (for hemo-diluted patients) and have a priming volume of about 200-225 ml. Such a filter would eliminate the need for pre-operative testing to determine a patient's prior sensitization, would eliminate protamine's adverse side effects which may lead to morbidity and/or death, and would reduce time and costs associated with adverse protamine reactions.